Weng L.,Jimei University |
Weng L.,Engineering Research Center for Aquatic Products Processing of Fujian Province |
Lin Y.,Jimei University |
Zhang L.,Jimei University |
And 6 more authors.
Journal of Chinese Institute of Food Science and Technology | Year: 2015
Prolyl endopeptidase (PEP) is a kind of endopeptidase which specifically cleaves low molecular weight peptides on the carboxyl side of proline residues, plays an important role in further degradation of collagens. In the present study, a prolyl endopeptidase was purified to homogeneity from grass carp muscle by ammonium sulfate fractionation, column chromatographies including DEAE-Sephacel, Phenyl-Sepharose and Hydroxyapatite and the biochemical characteristics were investigated. SDS-PAGE showed that the molecular weight of PEP was 75 ku. Optimal pH of the purified enzyme was 6.0, and it was stable at pH between 5.5 to 7.5. The optimal temperature of the enzyme was 30℃ while with poor thermal stability. The enzyme activity was strongly inhibited by SUAM, a competitive and specific inhibitor of prolyl endopeptidases. PEP showed it specifically cleaved fluorogenic substrates having proline residue on the carboxyl sides, strongly suggesting that enzyme is a kind of prolyl endopeptidase. The Km and kcat of the enzyme was 19.23 μmol/L and 2.5 s-1. MALDI-TOF-MS analysis revealed that the sequence of grass carp PEP shared 98.3%, 96.1% and 94.4% identities to PEPs from Oryzias latipes, Oreochromis niloticus and Danio rerio, respectively. ©, 2015, Chinese Institute of Food Science and Technology. All right reserved. Source